Microbiology Lab Media and Biochemical Tests Study Guide

Selective and Differential Media

Fundamental Concepts

Microbiological media are essential for culturing and identifying microorganisms. Selective and differential media are specialized types used to isolate and distinguish between different types of bacteria based on their growth and biochemical characteristics.

• Selective Media: Media that favor the growth of specific microorganisms while inhibiting others.

• Differential Media: Media that allow multiple organisms to grow but contain indicators to distinguish between them based on metabolic reactions.

• Defined Media: Media with known, specific chemical composition.

• Undefined (Complex) Media: Media containing complex ingredients (e.g., yeast extract) with unknown exact composition.

Key Terms:

• Fermentation: Anaerobic metabolic process converting sugars to acids, gases, or alcohol.

• By-products of Fermentation: Commonly include acids (e.g., lactic acid), gases (e.g., CO2), and alcohols (e.g., ethanol).

• pH Indicators: Chemicals that change color depending on the pH, used to detect acid or base production.

Example: Phenol red is a pH indicator that is yellow at acidic pH, red at neutral pH, and pink at alkaline pH.

Media and Biochemical Test Tables

Comparison of Common Selective and Differential Media

Media/Test	Selective Property	Differential Property	Indicator	Key Results
MSA (Mannitol Salt Agar)	High salt (7.5% NaCl) inhibits most bacteria except Staphylococcus spp.	Mannitol fermentation	Phenol red	Yellow = mannitol fermentation (+); Pink/red = no fermentation (-)
EMB (Eosin Methylene Blue Agar)	Dyes inhibit Gram-positive bacteria	Lactose fermentation	Eosin Y, Methylene blue	Dark purple/black = strong lactose fermenter; Pink = weak fermenter; Colorless = non-fermenter
HE (Hektoen Enteric Agar)	Bile salts inhibit Gram-positive bacteria	Lactose/sucrose/salicin fermentation, H2S production	Bromothymol blue, acid fuchsin, ferric ammonium citrate	Yellow/salmon = sugar fermentation; Black = H2S production

Additional info: Table entries inferred from standard microbiology lab media properties.

Biochemical Tests

Phenol Red Broth

Used to detect carbohydrate fermentation and gas production in bacteria.

• Carbon Source: Specific sugar (e.g., glucose, lactose)

• Nitrogen Source: Peptone

• Differential Properties: Fermentation of sugar, gas production

• pH Indicator: Phenol red

• Durham Tube: Small tube inside the broth to collect gas produced during fermentation

• Results:

  • Yellow broth: Acid production (fermentation, +)

  • Red/pink broth: No fermentation (-)

  • Gas bubble in Durham tube: Gas production (+)

Catalase Test

Detects the presence of the enzyme catalase, which breaks down hydrogen peroxide.

• Differential Property: Presence of catalase enzyme

• Indicator: Bubble formation upon addition of H2O2

• Reaction: $2H_2O_2 \rightarrow 2H_2O + O_2$

• Results:

  • Bubbles = catalase positive (+)

  • No bubbles = catalase negative (-)

• Interpretation: Catalase (+) is typical of aerobic and facultative anaerobic bacteria.

Oxidase Test

Identifies bacteria that produce cytochrome c oxidase, an enzyme of the electron transport chain.

• Differential Property: Presence of cytochrome c oxidase

• Indicator: Oxidase reagent (tetramethyl-p-phenylenediamine)

• Results:

  • Dark purple/blue color within 10-30 seconds = oxidase positive (+)

  • No color change = oxidase negative (-)

• Interpretation: Oxidase (+) is typical of aerobic bacteria.

Nitrate Broth Test

Tests for the ability of bacteria to reduce nitrate to nitrite or other nitrogenous compounds.

• Nitrogen Source: Potassium nitrate (KNO3)

• Differential Property: Nitrate reduction

• Chemical Indicators: Nitrate A & B reagents, zinc dust

• Results:

  • Red after A & B: Nitrite present (+)

  • No color after A & B, red after zinc: Nitrate not reduced (-)

  • No color after A & B and zinc: Nitrate reduced beyond nitrite (+)

Simmons Citrate Slant

Determines if an organism can use citrate as its sole carbon source.

• Carbon Source: Sodium citrate

• Nitrogen Source: Ammonium dihydrogen phosphate

• Differential Property: Citrate utilization

• Indicator: Bromothymol blue

• Results:

  • Blue slant: Citrate positive (+)

  • Green slant: Citrate negative (-)

• Inoculation: Needle used to avoid transferring excess carbon from loop.

Triple Sugar Iron (TSI) Agar

Used to differentiate bacteria based on glucose, lactose, and/or sucrose fermentation and sulfur reduction.

• Carbon Sources: Glucose, lactose, sucrose

• Nitrogen Source: Peptone

• Sulfur Source: Sodium thiosulfate

• Differential Properties: Sugar fermentation, gas production, H2S production

• pH Indicator: Phenol red

• Chemical Indicator: Ferrous sulfate (for H2S)

• Results:

  • Yellow butt/slant: Glucose and/or lactose/sucrose fermentation (+)

  • Red slant/yellow butt: Glucose fermentation only

  • Black precipitate: H2S production (+)

  • Gas bubbles/cracks: Gas production (+)

Motility and Indole Tests

Motility tests determine if bacteria can move through semi-solid media. Indole tests detect the breakdown of tryptophan to indole.

• Motility Indicator: Diffuse growth away from stab line

• Indole Test: Addition of Kovac's reagent; red ring indicates indole production (+)

• Enzyme: Tryptophanase breaks down tryptophan to indole

Summary Table: Key Biochemical Tests

Test	Enzyme/Process Detected	Positive Result	Negative Result
Catalase	Catalase	Bubbles	No bubbles
Oxidase	Cytochrome c oxidase	Dark purple/blue	No color change
Phenol Red Broth	Fermentation	Yellow (acid), gas bubble	Red/pink, no gas
Nitrate Broth	Nitrate reduction	Red after A&B, or no color after zinc	Red after zinc
Citrate	Citrate permease	Blue slant	Green slant

Additional info: Table entries inferred from standard test interpretations.